Molecular characterization and virulence of fungal pathogens associated with mass mortalities in hilsa Shad (Tenualosa ilisha)

The rising incidence of invasive microbial infections and the expanding spectrum of pathogens make early and accurate identification of the causative pathogen a daunting task. Accurate diagnosis enables the identification and characterization of microbes and offers new possibilities for the control and prevention of infectious diseases. For the first time, we isolated and characterized three fungal species, viz., Mucor circinelloides, Fusarium equiseti, and F. incarnatum from hilsa (Tenualosa ilisha). The characterization was based on fungus morphology (in vitro culture and microscopy), molecular (PCR, sequencing and phylogenetic analysis) and virulence features (survival assay, histological examination). The naturally infected fish exhibited significant histomorphology alterations in the gill, liver, kidney, spleen, and muscle tissue. Furthermore, the qPCR analysis, based on Pfaffl's relative standard curve method, exhibits significant up-regulation of Heat shock protein 70 (Hsp70), inducible nitric oxide synthase (iNOS) (except spleen) and tumour necrosis factor α (TNF-α) and Chemokine receptors (CCR) (only liver) were observed during a fungal infection in liver, kidney, and spleen tissue. In contrast, nuclear factor kappa B (NF-κB) and Interferon-gamma (IFN-γ) were significantly downregulated during infection. The fungal infection strongly influences the structures of the selected bacterial population (live bacterium)/cultivable bacterial enumeration and their abundance. Naturally infected fish were found to harbor Morganella morganii, Enterobacter cloacae, Enterococcus faecalis, and Proteus penneri. In contrast, non-infected fish contained Escherichia coli, Proteus mirabilis, Enterobacter cloacae, Enterococcus lactis, and Enterococcus gallinarum. The gut bacteria from infected hilsa displayed significantly higher (P-values < 0.05) haemolysin and swimming motility activity and biofilm production compared to those from non-infected fish. Additional research is required to clarify the virulence traits of the isolated fungi, as well as the impact of one or several isolated fungal species on the host's health, to assess the mortality risks posed by these fungi. Insights gained from the first cellular and molecular characterization of Mucor circinelloides, Fusarium equiseti, and F. incarnatum have provided valuable insights into disease epidemiology in hilsa farming.